Method of treating skin wounds with vectors encoding hepatocyte growth factor

ABSTRACT

The present invention relates to a therapeutic preventive agent that includes an angiogenic factor gene (such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hypoxia inducible factor (HIF)) as its active ingredient, and the administration of such an agent into the targeted skin diseases-affected area.

PRIORITY CLAIM

This is a § 371 U.S. national stage of PCT/JP02/04529, filed May 9,2002, and claims the benefit of Japanese Patent Application No.2001-139373, filed May 9, 2001.

TECHNICAL FIELD

The present invention relates to the use of an angiogenic factor genefor skin disease. More specifically, the present invention relates to atherapeutic or preventive agent comprising an angiogenic factor gene asthe active ingredient, and a method that comprises administering anangiogenic factor gene preventive to a target site. Examples ofangiogenic factors include hepatocyte growth factor (HGF) vascularendothelial growth factor (VEGF), fibroblast growth factor (FGF), andhypoxia inducible factor (HIF) Examples of skin diseases include wounds,alopecia (baldness), skin ulcers, decubitus ulcers (bedsores), scars(keloids), atopic dermatitis, and skin damage following skin grafts suchas autotransplantation and allotransplantation.

BACKGROUND ART

The expression “angiogenic factor” refers to a growth factor that notonly stimulates neovascularization and angiogenesis (initiated alongwith the activation of parent blood vessel endothelial cells) in vivo,but is also mitogenic for endothelial cells in vitro. Examples ofangiogenic factors include HGF, VEGF, FGF, and HIF. The firsttherapeutic application of angiogenic factors was reported by Folkman etal. (N. Engl. J. Med. 285, 1182-1186 (1971)). In later studies, the useof recombinant angiogenic factors such as the FGF family (Science 257,1401-1403 (1992); Nature 362, 844-846 (1993)) and VEGF was confirmed aspromoting and/or accelerating development of the collateral circulatorytract in animal models of myocardial and hind limb ischemia (Circulation90, II-228-II-234 (1994)). Furthermore, the present inventors discoveredthat HGF, like VEGF, functions as an endothelium-specific growth factor(J. Hypertens. 14, 1067-1072 (1996)).

HGF is a cytokine discovered to be a powerful growth-promoting factorfor mature stem cells, and its gene has been cloned (Biochem. Biophys.Res. Commun. 122: 1450(1984); Proc. Natl. Acad. Sci. USA. 83:6489(1986); FEBS Letters 22: 231(1987); Nature 342: 440-443(1989); Proc.Natl. Acad. Sci. USA. 87: 3200(1991)). HGF is a plasminogen-related andmesenchymer-derived pleiotropic growth factor, and is known to regulatecell growth and cell motility in various types of cells (Nature 342:440-443(1989); Biochem. Biophys. Res. Commun. 239: 639-644(1997); J.Biochem. Tokyo 119: 591-600(1996)). It is also an important factor inregulating blastogenesis and morphogenic processes during theregeneration of several organs. For example, HGF is a strong mitogen forepidermal cells such as hepatocytes and keratinocytes (Exp. Cell Res.196:114-120(1991)). HGF stimulates angiogenesis, induces celldissociation, and initiates endothelial cell movement (Proc. Natl. Acad.Sci. USA. 90: 1937-1941(1993); Gene Therapy 7: 417-427(2000)) Laterstudies revealed that HGF not only functions in vivo as a hepaticregeneration factor in the repair and regeneration of the damaged liver,but also has an angiogenic effect and plays an important role in thetherapy for or prevention of ischemic and arterial diseases (Symp. Soc.Exp. Biol., 47 cell behavior 227-234(1993); Proc. Natl. Acad. Sci. USA.90: 1937-1941(1993); Circulation 97: 381-390(1998)). There are reportsthat administration of HGF to rabbit hind limb ischemia models showedremarkable angiogenesis, improved blood flow, suppression of decrease inblood pressure, and improvement of ischemic symptoms. As a result ofthese reports, HGF is now considered to be expressed as an angiogenicfactor and to function as such.

As its name indicates, HGF was discovered in the liver. However, itactually exists throughout the entire body and has a cell-proliferatingaction. The vigorous cell division that occurs around an injury torepair the wound is also due to the action of HGF. The dermatology teamat Juntendo University discovered that HGF is also a hair growth factor.HGF promotes hair growth by promoting division of hair matrix cells.Administration of HGF to hair matrix cells on scalps which showprogressed androgen-related hair thinning is likely to regenerate thickhair.

Furthermore, HGF-induced angiogenesis in rat hearts with non-infarctedand infarcted myocardium (Proc. Natl. Acad. Sci. USA 90:8474-8478(1993)), and in rat corneas (Proc. Natl. Acad. Sci. USA 90:1937-1941(1993)) has been found in vivo.

Thus, HGF has a multitude of functions, not least of which is itsfunction as an angiogenic factor. Various attempts have been made toutilize HGF in pharmaceutical agents, however, HGF's half-life in theblood has made this a problem. HGF's short half-life of about tenminutes makes maintenance of its blood concentration difficult. Thus,translocation of an effective HGF dose to an affected area isproblematic.

VEGF is a dimeric glycoprotein that is mitogenic for endothelial cellsand can enhance vascular permeability. VEGF's mitogenic effect is directand specific to endothelial cells (Biochem. Biophys. Res. Commun., 161,851-858(1989)).

HIF promotes the production of erythrocytes and stimulates angiogenesisand erythropoietin (which increases oxygen supplied to the entire body).HIF is also the main transcription factor in the transcriptionalactivation of VEGF (which increases local oxygen supply), VEGF'sreceptor, and the genes for various enzymes involved in the glycolyticpathway (which provides resistance to cells by synthesizing ATP inanoxic conditions). HIF-1 is a heterodimer comprising HIF-1α and HIF-1β.HIF-1β (also called Arnt) also forms a heterodimer with the Ah receptor(which is associated with the metabolism of foreign substances such asdioxin) to function in the transcriptional regulation ofdrug-metabolizing enzyme genes.

In general, gene therapy can be used to treat various recovered clinicaldiseases (Science 256: 808-813(1992); Anal. Biochem. 162:156-159(1987)). Selection of an appropriate vector for gene transfer isparticularly important for successful gene therapy. Viruses,adenoviruses in particular, have been the preferred vectors for genetransfer. However, viral vectors are potentially dangerous when viralinfection-associated toxicity, lowered immunity, and mutagenic orcarcinogenic effects are considered. An example of an alternative methodfor gene transfer is the HVJ-liposome-mediated method, which has beenreported to be effective in vivo. This method uses liposomes incombination with a viral envelope, and shows hardly any toxicity(Science 243: 375-378(1989); Anal. NY Acad. Sci. 772: 126-139(1995)). Ithas been successfully used for in vivo gene transfer into tissuesincluding the liver, kidney, vascular wall, heart, and brain (GeneTherapy 7: 417-427(2000); Science 243: 375-378(1989); Biochem. Biophys.Res. Commun. 186: 129-134(1992); Proc. Natl. Acad. Sci. USA. 90:8474-8478(1993); Am. J. Physiol. 271(Regulatory Integrative Comp.Physiol. 40): R1212-R1220(1996)).

Wound healing comprises a succession of events including inflammation,angiogenesis, matrix synthesis, and collagen deposition, leading tore-endothelization, angiogenesis, and formation of granulation tissues(Clark R A F, “Overview and General Consideration of Wound Repair. TheMolecular and Cellular Biology of Wound Repair.” Plenum Press. New York(1996)3-50; Annu. Rev. Med. 46: 467-481(1995); J. Pathol. 178:5-10(1996)). These healing processes are regulated by a number ofmitogens and chemotactic factors, including growth factors such asfibroblast growth factor (FGF), transforming growth factor-α (TGF-α),transforming growth factor-β (TGF-β), epidermal growth factor (EGF),platelet-derived growth factor (PDGF), and vascular endothelial growthfactor (VEGF) However, few studies have focused on the effect of HGF onwound healing (Gastroenterology 113: 1858-1872(1997)).

Although there are several reports on the transfer of genes such as IGF,PDGF, and EGF into wounds (Gene Therapy 6: 1015-1020(1999) Lab. Invest.80: 151-158(2000); J. Invest. Dermatol. 112: 297-302(1999); Proc. Natl.Acad. Sci. USA 91: 12188-12192(1994)) none of these reports focus on thequantitative and qualitative changes in the number of factors involvedin wound healing, or on histopathological effects after HGF genetransfer.

Re-epithilization of a wound occurs by translocation of keratinocytesfrom the edges of the wound toward its center. In vitro, HGF enhancesproliferation, cell growth, and DNA synthesis in keratinocytes culturedunder physiological Ca²⁺ conditions (Exp. Cell Res. 196: 114-120(1991)). Furthermore, due to enhanced cell turnover, HGF has been foundto promote epithelial wound resealing in T84 intestinal monolayers (J.Clin. Invest. 93:2056-2065(1994)). In vivo administration of recombinantHGF has been found to promote regeneration of epithelial cells in ratkidneys damaged by anti-tumor agents (Gene Therapy 7: 417-427(2000)).However, in gastric ulcers produced in rats by cryoinjury, subcutaneousadministration of recombinant HGF had no effect on the ulcer-healingrate, despite the increase of human HGF concentration in the serum.Epithelial cell proliferation increased in the borders of the ulcerseight to 15 days after cryoinjury (Gastroenterology 113:1858-1872(1997)).

Transient upregulation of TGF-β expression is an important event inwound healing. TGF-β stimulates fibroblasts to produce matrix proteins,matrix protease inhibitors and integrin receptors, thereby modulatingmatrix formation and intercellular interactions at the wound site(Rokerts A B, Aporn M B: “Transforming growth factor-β. The Molecularand Cellular Biology of Wound Repair” Second Edition, by Clark R A F(Plenum Press. New York, 1996, 275-308)). Abnormal regulation andsustained overexpression of TGF-β1 would presumably contribute to anenhancement of tissue fibrosis, because increased expression of TGF-β1mRNA has been reported in tissues of patients with cutaneous fibrosis(for example, hypertrophic scars and keloids) (Am. J. Pathol. 152:485-493(1998)). Furthermore, TGF-β neutralizing antibodies not onlyreduced the cells in the wound granulation tissue of an adult wound, butalso improved the architecture of the neodermis (Lancet 339:213-214(1992)).

Proteinaceous formulations are generally administered intravenously. HGFhas been administered in ischemic disease models both intravenously andintra-arterially (Circulation 97: 381-390(1998)). Such intravenous orintra-arterial administrations of HGF to animal models have revealedHGF's effectiveness on ischemic or arterial diseases. However, as yet,no conclusion has been reached with regard to a specific and effectivemethod for administration, effective dose, and such. This isparticularly so in the case of the HGF protein, due to theabove-mentioned problems with half-life and transfer to the affectedarea. Thus, to date there has been no conclusion regarding an effectivemethod of administration, effective dose, etc.

DISCLOSURE OF THE INVENTION

An objective of the present invention relates to a therapeutic orpreventive agent for skin diseases that uses an angiogenic factor gene,and the use of these pharmaceutical agents.

The present inventors considered that HGF, which is an angiogenicfactor, might promote epithelial repair and angiogenesis during woundhealing. The present inventors investigated (i) whether, following genetransfer, human HGF mRNA and protein might distribute and deposit withinfull-thickness of wounds, (ii) whether the genetically transferredprotein might be biologically active, and (iii) whether the transferredprotein might have a biological effect on pathological conditions (forexample, mitogen activity involving several cells within full-thicknessof wounds, as well as re-epithelization in granulation tissues,angiogenesis, and deposition of the extracellular matrix, etc.).

Changes in wound tissues were also investigated to determine whetherthey related to TGF-β1 secretion. Measurements were made of the woundarea, the concentration of human and rat HGF protein in wound tissueafter HGF gene transfer, and the expression of the mRNA of otherconstitutive factors thought to be involved in wound healing such asTGF-β1, collagen type I (Colα2 (I)), collagen type III (Colα1 (III))desmin, and vascular smooth muscle α-actin (α-sm-actin) Asemiquantitative reverse transcription-polymerase chain reaction(RT-PCR) was used for these measurements. Morphogenic changes in thewound were investigated by in situ hybridization and immunohistochemicalmethods.

With these results, the present inventors demonstrated that directadministration of an angiogenic factor gene to a skin diseases-affectedarea is extremely effective. Specifically, it was found that in skinwounds, administration of an angiogenic factor gene yields effectiveresults.

Because therapy with an angiogenic factor gene is non-invasive, the genecan be administered any number of times depending on the condition ofthe disease.

Specifically, the subject matter of this invention is as follows:

-   (1) a therapeutic or preventive agent for skin diseases comprising    an angiogenic factor gene as the active ingredient;-   (2) the therapeutic or preventive agent according to (1) wherein the    angiogenic factor gene is an HGF gene, VEGF gene, FGF gene, or HIF    gene;-   (3) the therapeutic or preventive agent according to (1), wherein    the skin diseases is a wound, alopecia (baldness), skin ulcer,    decubitus ulcer (bedsore), scar (keloid), atopic dermatitis, or skin    damage following a skin graft including autotransplantation and    crosstransplantation;-   (4) the therapeutic or preventive agent according to (1) or (2),    wherein said therapeutic or preventive agent is in the form of a    tablet, pill, sugar-coated agent, capsule, liquid preparation, gel,    ointment, syrup, slurry, or suspension;-   (5) the agent according to any one of (1) to (3), wherein said agent    is used for transferring a gene into a cell by employing liposome    entrapment, electrostatic liposomes, HVJ-liposomes, improved    HVJ-liposome, viral envelope vectors, receptor-mediated gene    transfer, transfer of DNA into a cell using a particle gun (gene    gun) direct introduction of naked-DNA, DNA transfer into a cell by    ultrasonication, electroporation, or introduction using a positively    charged polymer;-   (6) a method for treating or preventing skin diseases, wherein the    method comprises introduction of an angiogenic factor gene into a    mammal; and-   (7) use of an angiogenic factor gene for producing a therapeutic or    preventive agent for skin diseases.

The term “angiogenic factor gene” used in the present invention refersto a gene that can express an angiogenic growth factor. Herein, the term“angiogenic factor” refers to a growth factor that has not only beenshown to stimulate in vivo neovascularization and angiogenesis(initiated along with activation of endothelial cells of the parentblood vessel), but has also been shown to be mitogenic for endothelialcells in vitro. Examples of the factor include HGF, VEGF, FGF, and HIFdescribed hereinafter.

In the present invention, the term “HGF gene,” as employed herein,refers to a gene that can express HGF (HGF protein). Specifically, thegene includes HGF cDNA (such as that described in Nature, 342, 440(1989), Japanese Patent Publication No. 2577091, Biochem. Biophys. Res.Commun., 163, 967 (1989), Biochem. Biophys. Res. Commun., 172: 321(1990)) where incorporated into appropriate expression vectors (e.g.non-viral vectors, viral vectors), such as those mentioned below. Thenucleotide sequence of the cDNA encoding HGF is described in theaforementioned literature. The sequence is also registered in databasessuch as Genbank. Thus, by using DNA segments appropriate to the DNAsequence as PCR primers, HGF cDNA can be cloned in an RT-PCR reaction,using, for example, mRNA derived from liver or leukocytes. This cloningcan be readily performed by one skilled in the art by referring to textssuch as Molecular Cloning Second Edition, Cold Spring Harbor LaboratoryPress (1989).

The term “VEGF gene,” as employed herein refers to a gene that canexpress VEGF (VEGF protein). Specifically, such a gene is exemplified byVEGF cDNA incorporated into appropriate expression vectors (e.g.non-viral vectors, viral vectors) such as those mentioned below. Due toselective splicing during transcription, there are four subtypes of theVEGF gene in humans (VEGF121, VEGF165, VEGF189, and VEGF206) (Science,219, 983 (1983); J. Clin. Invest., 84, 1470 (1989); Biochem. Biophys.Res. Commun., 161, 851 (1989)). Any of these VEGF genes can be used inthe present invention. However, the VEGF165 gene is preferred as itsbiological activity is the strongest of the VEGF genes. The VEGF genecan also be readily cloned by one skilled in the art, based on thesequences described in the literature (Science, 246, 1306 (1989)) andthe sequence information registered in databases. Modification of theVEGF gene can also be easily carried out.

The terms “FGF gene” and “HIF gene” as employed herein refer to genesthat can express FGF and HIF respectively. Such genes are exemplified bygenes incorporated into appropriate expression vectors (e.g. non-viralvectors, viral vectors) such as those mentioned below. Such genes canalso be readily cloned by one skilled in the art, based on the sequencesdescribed in known literature and sequence information registered indatabases. Modifications of these genes can also be easily carried out.

The angiogenic factor gene of the present invention is not limited tothose mentioned above. So long as the protein expressed by the gene iseffective as an angiogenic factor, the gene can be used as theangiogenic factor gene of the present invention. More specifically, theangiogenic factor gene of the present invention encompasses: 1) DNA thathybridizes under stringent conditions to the aforementioned cDNA; 2) DNAencoding a protein with the amino acid sequence of the protein encodedby the aforementioned cDNA, wherein one or more (preferably several)amino acids are substituted, deleted, and/or added; and such, so long asthe DNA encodes a protein which is effective as the angiogenic factor ofthis invention. The DNA described above in 1) and 2) can be readilyobtained, for example, by employing site-directed mutagenesis, PCR(Current Protocols in Molecular Biology edit., Ausubel et al. (1987)Publish. John Wiley & Sons Section 6.1-6.4), conventional hybridization(Current Protocols in Molecular Biology edit., Ausubel et al. (1987)Publish. John Wiley & Sons Section 6.3-6.4), etc.

Specifically, those skilled in the art can isolate DNA that hybridizeswith a DNA described above by using the above-mentioned angiogenicfactor gene or part thereof as a probe, or by using as a primer anoligonucleotide which specifically hybridizes with the angiogenicfactor. Typical stringent hybridization conditions for isolating DNAencoding a protein functionally equivalent to the angiogenic factor arethose of “1×SSC, 37° C.” or the like; more stringently, those of“0.5×SSC, 0.1% SDS, 42° C.” or the like; much more stringently, those of“0.1×SSC, 0.1% SDS, 65° C.” or the like. As the hybridization conditionsbecome more stringent, DNA more homologous to the probe sequence can beisolated. However, the above combinations of SSC, SDS, and temperatureare only examples, and those skilled in the art can achieve stringenciesequivalent to the above by appropriately combining these or otherconditions that determine hybridization stringency (probe concentration,probe length, time of reaction, etc.).

When compared to proteins of known angiogenic factor, proteins encodedby genes isolated using hybridization or PCR typically demonstrate highhomology at the amino acid level. The term “high homology” meanssequence homology of at least 50% or more, preferably 70% or more, morepreferably 90% or more (for example, 95% or more). The identity of aminoacid and nucleotide sequences can be determined using the BLASTalgorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA90:5873-5877, 1993). Based on this algorithm, programs such as BLASTNand BLASTX have been developed (Altschul et al. J. Mol. Biol. 215:403-410, 1990). When nucleotide sequences are analyzed by BLAST-basedBLASTN, the parameters are set, for example, as follows: score=100; andwordlength=12. Alternatively, when amino acid sequences are analyzed byBLAST-based BLASTX, the parameters are set, for example, as follows:score50; and wordlength=3. When BLAST and the Gapped BLAST program areused for the analysis, default parameters are used in each program. Thespecific techniques used in these analysis methods are already known(see, for example, the National Center for Biotechnology Information website).

The following describes methods, forms, and amounts of gene transferwhen gene therapy is employed as per the present invention.

When a gene therapy agent with the HGF gene as its active ingredient isadministered to a patient, the form of administration can be classifiedinto two groups: that using a non-viral vector, and that using a viralvector. Methods for the preparation and administration of these vectorsare described in detail in experiment manuals (Jikken Igaku(Experimental Medicine) Supplementary Volume, “Idenshichiryo noKisogijyutsu (Fundamental Techniques for Gene Therapy)”, Yodosha, 1996;Jikken Igaku (Experimental Medicine) Supplementary Volume, “Idenshidonyu& Hatsugenkaiseki Jikkenho (Experimental Methods for Gene Transfer &Expression Analysis)”, Yodosha, 1997; “Idenshi-chiryo Kaihatsu KenkyuHandbook (Handbook of Gene Therapy Research and Development)”, NihonIdenshichiryo Gakkai (The Japan Society of Gene Therapy) Edition, NTS,1999). Detailed explanations are given below.

A. Use of Non-Viral Vectors

A recombinant vector (where the target gene has been inserted into aconventional gene expression vector) can be used to insert a target geneinto cells and tissues as per the methods below.

Examples of methods for gene transfer into cells include: lipofection,calcium phosphate co-precipitation, the use of DEAE-dextran, directinfusion of DNA using a glass capillary tube, electroporation, etc.

Methods for gene transfer into tissues include the use of: internal typeliposomes, electrostatic type liposomes, HVJ (hemagglutinating virus ofJapan)-liposomes, improved type HVJ-liposomes (HVJ-AVE liposomes), viralenvelope vectors, receptor-mediated transfer, gene guns (the use of aparticle gun to import a carrier such as metal particles along withDNA)., direct introduction of naked-DNA, positively charged polymers,ultrasonic irradiation, etc.

The aforementioned HVJ-liposome is constructed by incorporating DNA intoa liposome formed by a lipid bilayer, then fusing this liposome with aninactivated Sendai virus (hemagglutinating virus of Japan: HVJ). The useof HVJ-liposomes is characterized by extremely high cell membrane fusioncompared to conventional liposome methods, and is the preferred form ofintroduction. Methods of preparing HVJ-liposomes have been described indetail (Experimental Medicine Supplementary Volume, “Idenshichiryo noKisogijyutsu (Fundamental Techniques of Gene Therapy)”, Yodosha, 1996;Experimental Medicine Supplementary Volume, “Idenshidonyu &Hatsugenkaiseki Jikkenho (Experimental Methods for Gene Transfer &Expression Analysis)”, Yodosha (1997); J. Clin. Invest. 93:1458-1464(1994); Am. J. Physiol. 271: R1212-1220(1996), etc.). Use ofthe HVJ-liposome in transfection also includes, for example, the methodsdescribed in Molecular Medicine 30: 1440-1448(1993); ExperimentalMedicine, 12: 1822-1826(1994); Protein, Nucleic Acid, and Enzyme, 42,1806-1813(1997); and preferably includes the method described inCirculation 92(Suppl. II): 479-482(1995).

The Z strain (available from ATCC) is the preferred HVJ strain, howeverin essence, other HVJ strains (for example, ATCC VR-907, ATCC VR-105,etc.) may be used.

Herein, the term “viral envelope vector” refers to a vector thatincorporates a foreign gene into a viral envelope. Viral envelopevectors are gene transfer vectors in which the viral genome has beeninactivated. Since viral proteins are not produced, the vector is safeand its cytotoxicity and antigenicity are low. By incorporating a geneinto such a viral envelope vector (e.g. one that uses an inactivatedvirus), a highly efficient gene transfer vector that is safe for usewith cultured cells and biological tissues can be prepared. Viralenvelope vectors can be prepared, for example, using the methoddescribed in WO 01/57204 (PCT/JP01/00782). Examples of viruses used toprepare gene transfer vectors include both wild-type viruses andrecombinant viruses, and such examples include Retroviridae,Togaviridae, Coronaviridae, Flaviviridae, Paramyxoviridae,Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Poxyiridae,Herpesviridae, Baculoviridae, and Hepadnaviridae. A viral envelopevector using HVJ is particularly suitable. Furthermore, a gene transfervector can be prepared using a recombinant Sendai virus, as described byHasan, M. K. et al. (Journal of General Virology, 78, 2813-2820 (1997))or Yonemitsu, Y. et al. (Nature Biotechnology 18, 970-973 (2000)).

Direct transfer of naked-DNA is the most convenient of the methodsmentioned above, and is thus the preferred method of introduction.

With respect to the present invention, any expression vector can be usedso long as it can express the desired gene in vivo, and includes, forexample, pCAGGS (Gene, 108, 193-200 (1991)), pBK-CMV, pcDNA3.1, orpZeoSV (Invitrogen, Stratagene).

B. Use of Viral Vectors

Viral vectors such as recombinant adenoviruses and retroviruses aretypically used. More specifically, a desired gene is introduced into aDNA or RNA virus, such as an avirulent retrovirus, adenovirus,adeno-associated virus, herpes virus, vaccinia virus, poxvirus,poliovirus, Sindbis virus, Sendai virus, SV40, or immunodeficiency virus(HIV). The recombinant virus is then infected into the cell, thusintroducing the desired gene.

Of the viral vectors mentioned above, the infection efficiency ofadenoviruses is known to be much higher than other viral vectors. Thus,the use of the adenovirus vector system is preferred.

Methods for introducing an agent of the present invention during genetherapy include: (i) in vivo introduction of a gene therapy agentdirectly into the body; and (ii) ex vivo introduction of a gene therapyagent into a cell harvested from the body, followed by reintroduction ofthe modified cell into the body (Nikkei Science, April 1994, 20-45;Gekkann Yakuji 36 (1), 23-48, 1994; Jikken Igaku (Experimental Medicine)Supplementary Volume, 12 (15), 1994; “Idenshi-chiryo Kaihatsu KenkyuHandbook (Handbook of Gene Therapy Research and Development)”, NihonIdenshichiryo Gakkai eds. (The Japan Society of Gene Therapy) Edition,NTS, 1999). The in vivo method is preferred in the present invention.

Various formulations (for example, liquid preparations) suitable foreach of the above-mentioned methods of administration may be adopted asthe form of the preparation. For example, an injection containing a geneas the active ingredient can be prepared by conventional methods whichmight include dissolving a gene in an appropriate solvent (e.g. a buffersolution, such as PBS, physiological saline, and sterilized water);sterilizing by filtration as necessary, and then loading into a sterilecontainer. Conventional carriers and such like may be added to theinjection as required. Alternatively, liposomes such as the HVJ-liposomecan be prepared as suspensions, frozen agents, or centrifugallyconcentrated frozen agents.

For skin diseases, a therapeutic or preventive agent of this inventionmay be locally administered to the affected area of the skin, preferablyin the form of an ointment. This ointment is an entirely homogenoussemi-solid external agent with a firmness appropriate for easyapplication to the skin. Such an ointment normally includes fats, fattyoils, lanoline, Vaseline, paraffin, wax, hard ointments, resins,plastics, glycols, higher alcohols, glycerol, water or emulsifier and asuspending agent. Using these ingredients as a base, a decoy compoundcan be evenly mixed. Depending on the base, the mixture may be in theform of an oleaginous ointment, an emulsified ointment, or awater-soluble ointment oleaginous ointments use bases such as plant andanimal oils and fats, wax, Vaseline and liquid paraffin. Emulsifiedointments are comprised of an oleaginous substance and water, emulsifiedwith an emulsifier. They may take either an oil-in-water form (O/W) or awater-in-oil-form (W/O). The oil-in-water form (O/W) may be ahydrophilic ointment. The water-in-oil form (W/O) initially lacks anaqueous phase and may include hydrophilic Vaseline and purifiedlanoline, or it may contain a water-absorption ointment (including anaqueous phase) and hydrated lanoline. A water-soluble ointment maycontain a completely water-soluble Macrogol base as its main ingredient.

A pharmaceutically acceptable and preferable carrier is Vaselinecontaining 5% stearyl alcohol, or Vaseline alone, or Vaseline containingliquid paraffin. Such carriers enable pharmaceutical compositions to beprescribed in forms appropriate for patient consumption, such astablets, pills, sugar-coated agents, capsules, liquid preparations,gels, ointments, syrups, slurries, and suspensions.

Alternatively, when locally administered into cells in an affected areaor a tissue of interest, a therapeutic or preventive agent of thisinvention may contain a synthetic or natural hydrophilic polymer as thecarrier. Examples of such polymers include hydroxypropyl cellulose andpolyethylene glycol. A gene or vector of the present invention is mixedwith a hydrophilic polymer in an appropriate solvent. The solvent isthen removed by methods such as air-drying, and the remainder is thenshaped into a desired form (for example, a sheet) and applied to thetarget site. Formulations containing such hydrophilic polymers keep wellas they have a low water-content. At the time of use, they absorb water,becoming gels that also store well. In the case of sheets, the firmnesscan be adjusted by mixing a polyhydric alcohol with a hydrophilicpolymer similar to those above, such as cellulose, starch and itsderivatives, or synthetic polymeric compounds. Hydrophilic sheets thusformed can be used as the above-mentioned sheets.

Genes selected from angiogenic factor genes such as those used in thepresent invention (e.g. HGF, VEGF, FGF, HIF, etc.) may be used inmultiple combinations or alone. Furthermore, factors other than theangiogenic factors mentioned above, and which are known to have anangiogenic effect, may also be used in combination or alone. Forexample, factors such as EGF have been reported to have an angiogeniceffect, and such genes can be used. Furthermore, growth factors such asEGF have been reported to repair a variety of tissue cell injuries, andsuch genes may also be used.

Skin diseases according to the present invention includes wounds,alopecia (baldness), skin ulcers, decubitus ulcers (bedsores) scars(keloids), atopic dermatitis, and skin damage following skin grafts suchas autotransplantation and crosstransplantation. Preventive agent,according to the present invention, refers to a pharmaceutical agentwhich prevents the onset (or incidence) of the above-mentioned diseases,or a pharmaceutical agent which reduces symptoms caused by theabove-mentioned diseases, or a pharmaceutical agent which acceleratesamelioration of these symptoms. These preventive agents are alsoincluded in the present invention.

Herein, “alopecia” refers to the phenomenon of thinning hair, where thehair cycle becomes extremely short, such that even thick hair falls outmid-growth, and as a result, the hairs that do grow are soft, fine andshort. The expression “skin ulcers” means damage to deep tissues,reaching to the dermis or to the hypodermal tissue. Skin ulcers arecategorized into ischemic ulcers, congestive ulcers, diabetic ulcers,decubitus ulcers, radiation ulcers instillation leakages, etc.“Decubitus ulcers” refers to a pathological condition where necrosisoccurs by occlusion of a tissue's peripheral blood vessels due tocontinuous compression experienced at the contact surface of the body.Decubitus ulcers are intractable ulcers having a dry necrotic mass witha clear border, which form at sites of long-term compression, such asthe back of the head, the back, and the hips of bedridden people. “Scars(keloids)” occur after skin damage and means hypertrophy of theconnective tissue in which the wound surface produces a flat protrusionand sometimes forms claw-like projections. Some scars are proliferative,and continue to expand to the surrounding region, and beyond theoriginal wound site. Factors that cause an external wound to form akeloid include systemic factors (such as genetic factors, age, andhormonal factors), and local factors (such as susceptibility to scarsdepending on the part of the body). Scars are categorized intohyperplastic scars, keloid scars, true keloids, etc.

Administration sites and methods for gene therapy agents of the presentinvention are selected such that they are appropriate to the disease andsymptoms to be treated. The preferred administration method isparenteral administration. Furthermore, the preferred administrationsite is at the site of skin diseases. Herein, the term “site of skindiseases” refers to a site including the skin diseases-affected area andits surrounding region.

Specifically, administration to the skin diseases site can be carriedout intravascularly, intramuscularly, and such, as well as byadministration to surface layers with ointments and such Therefore, atthe sites of wounds, baldness, decubitus ulcers (bedsores), keloids,atopic dermatitis, and skin grafts such as autotransplantation andcrosstransplantation, angiogenesis in the affected area is enhanced, andblood flow is improved by intravascular and intramuscular administrationusing a syringe or catheter, or by surface application using an ointmentor such. In this way, the function of the affected area can be recoveredand normalized.

Application of an HGF gene of the present invention by active genetransfer allows treatment of wounds, baldness, skin ulcers, decubitusulcers (bedsores), scars (keloids), atopic dermatitis, and skin damagefollowing skin grafts such as autotransplantation andcrosstransplantation, and enables functional recovery in patients forwhom conventional therapeutic methods are not an appropriate option. Atherapeutic or preventive agent of the present invention contains anangiogenic factor gene in an amount sufficient to accomplish theobjectives intended by the pharmaceutical agent, i.e. it contains anangiogenesis gene in a “therapeutically effective amount” or a“pharmacologically effective amount”. A “therapeutically effectiveamount” or “pharmacologically effective amount” is an amount ofpharmaceutical agent required to produce the intended pharmacologicalresults, and is the amount required to relieve the symptoms of thepatient to be treated. Assays useful in confirming the effective dosefor a particular application include methods for measuring the degree ofrecovery from target diseases. The amount that should actually beadministered varies depending on the individual being treated, and ispreferably an amount optimized to achieve the desired effects withoutmarked side effects.

Therapeutically effective amounts, pharmacologically effective amounts,and toxicity can be determined by cell culture assays or optionally, byusing appropriate animal models. Such animal models can be used todetermine the desired concentration range and administration route forthe pharmaceutical agent. Based on these animal models, one skilled inthe art can determine the effective dose in a human. The dose ratio oftherapeutic effect to toxic effect is called the therapeutic index, andthis can be expressed as the ratio ED50:LD50. Pharmaceuticalcompositions with a large therapeutic index are preferred. Anappropriate dose is selected according to the dosage form, the patient'ssensitivity, age and other conditions, and the type and severity of thedisease. Although the dose of a therapeutic agent of the presentinvention differs depending on the condition of the patient, the adultdose of an HGF gene is in the range of approximately 1 μg toapproximately 50 mg, preferably in the range of approximately 10 μg toapproximately 5 mg, and more preferably from the range of approximately50 μg to approximately 5 mg.

A therapeutic agent of the present invention is preferably administeredonce every few days or few weeks, where the frequency of administrationis selected such that it is appropriate to the patient's symptoms. Acharacteristic of the therapeutic agent of the present invention isthat, due to its non-invasive administration, it can be administered anynumber of times depending on the symptoms.

With regards to the present invention, there are no restrictionsregarding the animal into which the angiogenic factor gene can betransferred, however mammals are preferred. Examples of mammals include,without limitation, humans, and non-human mammals such as monkeys, mice,rats, pigs, cows and sheep.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows human HGF concentration (A), and rat HGF concentration (B),in wounds after gene transfer. *, p<0.05 versus value for the controlrats. n=5 for each group. ND: not detected.

FIG. 2 is a set of photographs showing the distribution of human HGFmRNA (a to d) and protein (e to h) in HGF gene-transfer rats. Scale barsin the figure represent 100 μm in a and e, and 200 μm in b to d, f to h.

FIG. 3 shows the size of the wound area after gene transfer as apercentage of original wound area.

FIG. 4 is a graph (A), and a set of photographs (B), indicatingexpression of PCNA in the epidermis on the edge of the wound in ratsafter gene transfer. (A) shows the percentage of PCNA-positive cells inthe epidermis after gene transfer, and (B) is a photograph showing theexpression of PCNA. In the figure, a to c are photographs of HGFgene-transfer rats at days 3, 7, and 14 respectively, and d to f arephotographs of control rats at days 3, 7, and 14 respectively. Scalebars represent 200 μm in a to f.

FIG. 5 is a graph (A), and a set of photographs (B), showing expressionof PCNA in the granulation tissue of rats after gene transfer. (A) showsthe percentage of PCNA-positive cells in the granulation tissue aftergene transfer, and (B) shows the expression of PCNA. In the figure, a toc are photographs of HGF gene-transfer rats at days 3, 7, and 14respectively, and d to f are photographs of control rats at days 3, 7,and 14 respectively. Scale bars represent 200 μm in a to f.

FIG. 6 is a graph (A), and a set of photographs (B), showing themicrovessel count in the granulation tissue of rats after gene transferby immunohistochemistry for factor VIII. (A) shows the microvessel countin the granulation tissue, and (B) shows the result ofimmunohistochemistry for factor VIII. In the figure, a to c arephotographs of HGF gene-transfer rats at days 3, 7, and 14 respectively,and d to f are photographs of control rats at days 3, 7, and 14respectively. Scale bars represent 200 μm in a to f.

FIG. 7 shows the result of RT-PCR for Colα2 (I) mRNA. (A) is a real-timeamplification plot for Colα2 (I), obtained using a semi-quantitativeRT-PCR method. (B) is the standard curve for the threshold cycle ofRT-PCR.

FIG. 8 is a set of graphs and a photograph indicating the results ofRT-PCR for TGF-β1, Colα2 (I), α-actin, desmin, and Colα1 (III) mRNA. (A)shows the detection of PCR products, and (B) shows the results ofsemi-quantitative RT-PCR.

FIG. 9 shows the results of detecting hydroxyproline concentration inthe wounds of rats after gene transfer.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is specifically illustrated belowwith reference to Examples, but is not to be construed as being limitedthereto.

(1) HGF Protein Concentration in Wound Tissues and in Plasma

-   1. Laboratory animals Sixty-five male Wistar rats, approximately    eleven weeks old and weighing 310 g to 370 g, were assigned to one    of two experiments, and then housed two per cage in a    temperature-controlled room with a twelve-hour light-dark cycle. All    rats were given commercial feed and tap water ad libitum. This    experiment was performed in accordance with the Care and Use of    Laboratory Animals of the National Institute of Health protocol.    This protocol was approved by the Committee on the Ethics of Animal    Experiments in the National Defense Medical College.-   2. HGF expression vector Human HGF cDNA (2.2 kb) was inserted    between the EcoRI/NotI sites of the pUC-SRα expression vector    plasmid. In this plasmid, transcription of HGF cDNA is controlled by    the SRα promoter (Nature 342: 440-443(1989)).-   3. HVJ-liposomes (HMG)-1 (50 μl) purified from calf thymus was mixed    with plasmid DNA (200 μg) in a total volume of 200 μl of isotonic    solution (137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.6) at 20° C.    for one hour, and then this mixture was added to 10 mg of dry lipid    (a 1:4.8:2 mixture of phosphatidylserine, phosphatidylcholine, and    cholesterol). The liposome-DNA-HMG-1 complex suspension was mixed,    ultrasonicated for three seconds, and then shaken for 30 minutes to    form liposomes. Purified Sendai virus (HVJ) (Z-strain) was    inactivated by UV irradiation (110 erg/mm²/sec) for three minutes    immediately before use. The liposome suspension (0.5 mL, containing    10 mg of lipid) was mixed with HVJ (30,000 hemagglutinating units)    in a total volume of 3 mL of isotonic solution. After mixing, this    was incubated at 4° C. for ten minutes, and then at 37° C. for 30    minutes with mild shaking. Free HVJ was removed by sucrose density    gradient centrifugation. HVJ-liposome-DNA complex was collected from    the top layer, and used immediately.-   4. Wound tissues and blood samples Forty-one rats were divided into    two groups (the HGF gene-transfer group and the control vector    group) for biochemical and histological examination of wounds. The    rats were anesthetized by intra-peritoneal injection of sodium    pentobarbital (0.5 ml/kg), then the hair on their backs was shaved,    the skin cleaned, and a 14 mm deep wound was made on the back of    each animal. Three days later, these same rats (under pentobarbital    anesthesia) received a subcutaneous injection of either HVJ-liposome    (500 μl) containing 100 μg of HGF cDNA, or of a control vector.    These injections were delivered to the edge of each wound using a    27-G needle. At 3, 7, and 14 days after the injection, the animals    were decapitated under anesthesia, and blood samples were collected    for HGF determination. The blood samples were placed in chilled    tubes containing EDTA (2 mg/ml), and then centrifuged. The resultant    plasma was frozen immediately, and stored at −80° C. until analysis.    At autopsy, the rats' skins were removed. The tissues of five rats    per group were quantitatively determined and then cut in half. One    half was frozen in liquid nitrogen and stored at −80° C. until use.-   5. ELISA test Tissue samples from five rats in each group were    homogenized for one minute in four times their volume of 20 mM    Tris-HCl buffer (pH 7.5) containing 0.1% 2 M NaCl, 0.1% Tween-80, 1    mM PMSF, and 1 mM EDTA, using a polytron homogenizer (24,000 rpm;    Kinematica AG, Lucerne, Switzerland). The homogenate was centrifuged    at 15,000×g for 30 minutes at 4° C. The supernatant and pellet were    stored at −80° C. until carrying out an enzyme-linked    immunoabsorbent assay (ELISA) for HGF protein. Human HGF protein    concentration was measured with ELISA, using an anti-human-HGF    monoclonal antibody. Rat HGF concentration was also measured with    ELISA, using an anti-rat-HGF monoclonal antibody (Institute of    Immunology, Tokyo, Japan). The human HGF ELISA specifically detected    human HGF, but not rat HGF. Plasma HGF protein concentration was    measured in 50 L of rat plasma., using the above-mentioned ELISA.-   6. Results ELISA revealed that human HGF protein levels were greatly    increased at 3, 7, and 14 days after HGF gene transfer into the    wound tissues of the HGF gene-transfer rats, and were not detected    at all in the control rats (FIG. 1A). However, human HGF protein was    not detected in plasma samples derived from the HGF gene-transfer    rats. Rat HGF levels in the wound tissues of the HGF gene-transfer    rats significantly increased for the first time three days after    gene transfer (FIG. 1B, p<0.05).    (2) Expression of Human HGF mRNA and Protein in Wound Tissues

By a method similar to that described in (1), wounds were made on rats,and HVJ-liposome or control vector was administered, the animals weredecapitated under anesthesia at 3, 7., and 14 days after injection, andthe wound tissues thus obtained were fixed inperiodate-lysine-paraformaldehyde (PLP) solution.

-   1. In situ hybridization In situ hybridization of HGF was carried    out using deparaffinated 4% paraformaldehyde-fixed sections treated    with 0.2 N HCl for 20 minutes, then incubated in 2×SSC for ten    minutes at 37° C., and finally incubated in 5 μg/ml proteinase K for    ten minutes at 37° C. Each section was then fixed in ˜4%    paraformaldehyde for five minutes, and incubated for ten minutes in    0.1 mol/L of triethanolamine buffer (pH 8.0) containing 0.25%    (vol/vol) acetic anhydride to prevent non-specific binding due to    tissue oxidation. The full-length human HGF cDNA (which was inserted    between the EcoRI and NotI sites of the pUC-SRα expression vector    plasmid) was digested by restriction enzymes for EcoRI. The    resulting fragments of HGF cDNA (848 bp) were then ligated between    the EcoRI cloning sites of pGEM-7Zf(+) (Promega, Madison, Wis.). The    antisense probe and the corresponding sense probe were labeled with    digoxigenin using SP6 and T7 polymerase respectively, by means of a    RNA labeling kit (Boehringer Mannheim, Postfach, Germany).    Hybridization was performed overnight at 42° C. in 50% (vol/vol)    deionized formamide, 5× Denhardt's solution, 5% (weight/vol) dextran    sulfate, 2×SSC, 0.3 mg/ml salmon-sperm DNA, 5 mM EDTA, and 0.01    μg/ml digoxigenin-labeled probes. After performing a final    stringency wash at 55° C. for 20 minutes, hybridization was    immunologically detected.-   2. Immunohistochemistry The immunoperoxidase method was directly    applied to the deparaffinated sections. A mouse monoclonal antibody    against HGF was used (1:20; Institute of Immunology, Tokyo, Japan),    and a horseradish peroxidase-labeled secondary antibody against    rabbit immunoglobulin was used (Chemicon International Inc., 1:250    dilution). The mouse monoclonal antibody was specific to human HGF,    and not rat HGF.-   3. Results In the wound tissues of HGF gene-transfer rats, HGF mRNA    was detected three days after gene transfer in squamous cells in the    epithelium on the edge of the wound, in endothelial cells and smooth    muscle cells of blood vessels, and in fibroblasts in the granulation    tissues (FIG. 2 a, 2 b, 2 e, and 2 f). In contrast, HGF mRNA was not    detected at all in the control rats. Similarly, human HGF protein    was detected in the same cell types (squamous cells in the    epithelium, endothelial cells and smooth muscle cells of blood    vessels, and fibroblasts in the granulation tissues) of HGF    gene-transfer rats, and was not detected in the control rats (FIGS.    2 c and 2 g). HGF expression was subsequently maintained up to 14    days after gene transfer (the last day of examination) (FIGS. 2 d    and 2 h).-   (3) Wound Lesion Size

By a method similar to that described in (1), wounds were made on rats,and HVJ-liposome or control vector was administered. Following gene (orvector) transfer, the wound areas of 20 rats were measured.

-   1. Measurement of wound area Wound area was measured from tracings    taken at 0, 3, 7, 10, and 14 days after gene transfer, using an    image analyzer (TOSPIX-U, AS3260C, and image analysis package    software; Toshiba, Tokyo, Japan). Wound area was represented as a    percentage of the initial area (as measured on day zero after gene    transfer). The day of complete healing was taken to be the day that    the epithelium completely closed the full extent of the    full-thickness wound.    2. Results The wound lesion area (expressed as a percentage of the    original wound lesion area on day zero after gene transfer) was    significantly decreased in HGF gene-transfer rats (compared to    control rats) from three days after gene transfer (FIG. 3, p<0.05).    However, there was no difference between HGF gene transfer rats and    control rats in the days required for complete healing.-   (4) Cell Proliferation and Angiogenesis in Wounds

By a method similar to that described in (1), wounds were made on rats,HVJ-liposome or control vector was administered, the animal wasdecapitated under anesthesia at 3, 7, and 14 days after injection, andthe wound tissues obtained were fixed in PLP.

1. Proliferating Cell Nuclear Antigen (PCNA) Measurements

Expression of PCNA in each tissue was detected as an index of cellproliferation. The immunoperoxidase method was applied directly todeparaffinated epithelium and granulation tissues. This method usedmouse monoclonal antibody against PCNA (PC-10, 1:100, Dako Inc.,Glostrup, Denmark), and horseradish peroxidase-labeled secondaryantibody against rabbit immunoglobulin (Chemicon International Inc.,dilution 1:250). Autoclave pretreatment in 0.01 M of citrate buffersolution (pH 6.0) was performed for 15 minutes at 120° C. beforeimmunohistochemistry on PC-10. Incubation with a primary antibody wasomitted as the negative control. For the PC-10 analysis, and on thebasis of immunoreaction in at least 1,000 tumor cells, the percentage ofnuclei with a positive immunoreaction was determined (PCNA index).

-   2. Measurement of angiogenesis Wound microvessel counts were    evaluated by light microscopy in the areas containing the largest    number of blood vessels. The number of blood vessels was determined    in a continuous 200× field (20× objective lens and 10× ocular lens;    0.0925 mm² per field). Furthermore, in a similar manner to the    method described in (1), angiogenesis was measured by investigating    factor VIII in the epithelial cells of granulation tissues using a    polyclonal antibody against factor VIII (1:100, Dako Inc., Glostrup,    Denmark)-   3. Results PCNA indices in the epithelium on the edge of the wound    and in granulation tissues were both significantly increased in HGF    gene-transfer rats at three and seven days after gene transfer    (compared to control rats) (FIGS. 4 and 5, p<0.05). Microvessel    counts in the granulation tissues (as detected by    immunohistochemistry for factor VIII) were significantly increased    in HGF gene-transfer rats at three days after gene transfer (FIG. 6,    p<0.05).    (5) Expression of Dermal Components in the Wound

By a method similar to that described in (1), wounds were made on rats,HVJ-liposome or control vector was administered, and RNA was extractedfrom the wound tissues of animals decapitated under anesthesia at 3, 7,and 14 days after injection.

-   1. Total RNA Extraction and Semi-Quantitative RT-PCR

Semi-quantitative RT-PCR was used to examine the expression of variousmRNA in the skin tissue of five rats in each group (Lab. Invest 79:679-688(1999)). Total RNA in the skin tissues was isolated using acidguanidinium isothiocyanate-phenol-chloroform extraction and ethanolprecipitation (Anal. Biochem. 162: 156-159(1987)). RT-PCR was performedusing an amplification reagent kit (TaqMan EZRT-PCR kit; AppliedBiosystems, Alameda, Calif.) with several primers. The following primerswere prepared using an automated DNA synthesizer: TGF-β1, Colα2 (I),Colα1 (III), desmin, α-sm-actin, and glyceraldehyde-3-phosphatedehydrogenase (GAPDH). Table 1 shows the temperature conditions andsequence information for all of the PCR primers and TaqMan probes used(Hepatology 24: 636-642(1996)). TaqMan probes were labeled at the 5′-endwith a reporter dye molecule, FAM (6-carboxyfluorescein), and at the3′-end with a quencher dye, TAMRA (6-carboxytetramethylrhodamine). Thereaction master mix was prepared according to the manufacturer'sprotocol, giving final concentrations of 1× reaction buffer, 300 μMdATP, 300 μM dCTP, 300 μM dGTP, 600 μM dUTP, 3 mM Mg(OAc)₂, 0.1 U/μlrTth DNA polymerase, 0.01 U/μl AmpErase UNG, 900 nM primers, and 200 nMTaqMan probe. The RT reaction solution was incubated at 60° C. for 30minutes, then at 95° C. for five minutes to inactivate AmpErase UNG. PCRwas performed using an ABI PRISM 7700 Sequence detector (AppliedBiosystems) During each PCR cycle, the TaqMan probe was cleaved by the5′->3′ exonuclease activity of rTth DNA polymerase, thereby increasingreporter dye fluorescence at the appropriate wavelength. The increase influorescence was proportional to the concentration of template in thePCR (FIG. 7A). Threshold fluorescence was set at 6.965 times thestandard deviation of the average value obtained from the controlwithout the template (following the protocol of the TaqMan RT-PCR kit).A standard curve was obtained using the threshold cycle established foreach RNA level using four separate wells (FIG. 7B) PCR products wereseparated by electrophoresis in a 3% agarose gel, and stained withethidium bromide (FIG. 8A).

TABLE 1 Sense Antisense Annealing Size of primer primer TaqMan probetemperature product mRNA (5′-3′) (5′-3′) (5′-3′) (C.) Cycle (bp) GAPDHCTTCACCACC GGCATGGACT CCTGGCCAAGG 60 40 238 ATGGAGAAGG GTGGTCATGATCATCCATGAC C G AACTTT SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 TGF-β1TGAGTGGCTG GCAGTTCTTC CAGTGGCTGAA 64 50 301 TCTTTTGACG TCTGTGGAGCCCAAGGAGACG TC TG GAAT SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 Colα2(I)GGCTGCTCCA CCAGAGGTGC ATACAAAACGA 60 40 97 AAAAGACAAA AATGTCAAGGATAAGCCATCT TG AA CGCCTGCC SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9Colα1(III) GTGAAAGAGG GAGTTCAGGG TGCTGCCATTG 64 50 302 ATCTGAGGGCTGGCAGAATT CTGGAGTTGGA TC T SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12α-sm-actin CGATAGAACA GCATAGCCCT AACTGGGACGA 60 50 301 CGGCATCATCCATAGATAGG CATGGAAAAGA AC CA TCTGG SEQ ID NO:13 SEQ ID NO:14 SEQ IDNO:15 Desmin AGCGCAGAAT TGTCGGTATT CTCAGGGACAT 60 50 301 TGAGTCACTCCCATCATCTC CCGTGCTCAGT AA CT ATGAGA SEQ ID NO:16 SEQ ID NO:17 SEQ IDNO:18 GAPDH, rat glyceraldehyde-3-phosphate dehydrogenase; TGF-β1, rattransforming growth factor-β1; Colα2(I), rat α-2 type I collagen,segment 2; Colα1(III), rat collagen type III α-1; desmin, rat desmin;α-sm-actin, rat vascular smooth muscle α-actin.2. Results Expression of desmin mRNA in the wound tissues of HGFgene-transfer rats was significantly increased (compared to controlrats) at three days after gene transfer, as determined bysemi-quantitative RT-PCR. Expression of TGF-β1 and Colα2 (I) mRNA wassignificantly decreased at 7 and 14 days after gene transferrespectively (FIG. 8B, p<0.0.5).(6) Hydroxyproline Concentration in the Wound

By a method similar to that described in (1)., wounds were made on rats,HVJ-liposome or control vector was administered, and tissue samples wereprepared from wound tissues obtained from animals decapitated underanesthesia at 3, 7, and 14 days after injection.

-   1. ELISA test The samples were homogenized for one minute in four    times their volume of 20 mM Tris-HCl buffer (pH 7.5) containing 0.1%    2 M NaCl, 0.1% Tween-80, 1 mM PMSF, and 1 mM EDTA, using a polytron    homogenizer (24,000 rpm; Kinematica AG, Lucerne, Switzerland). The    homogenate was centrifuged at 15,000×g for 30 minutes at 4° C., and    the pellet was hydrolyzed in 6 N HCl at 110° C. for 16 hours.    Hydroxyproline content was determined using an amino acid analyzer    (Model 835; Hitachi Ltd. Tokyo, Japan).-   2. Results At 3, 7, and 14 days after gene transfer, hydroxyproline    concentration in the wounds of HGF-transfer rats was significantly    lower than in the wounds of control rats (FIG. 9, p<0.05).

Each of the results obtained in the Examples above are expressed as amean±standard deviation of the mean (SEM). When a significant F-valuewas obtained by analysis of variance (ANOVA), Fisher's protectedleast-significant-difference test was applied. p<0.05 was taken to be asignificant difference.

INDUSTRIAL APPLICABILITY

Herein, an increased amount of human and rat HGF protein in the woundtissues of HGF gene-transfer rats, when compared to control rats, wasobserved using ELISA and immunohistochemistry. Similarly, usingimmunohistochemistry, the wound tissues of HGF gene-transfer rats wereobserved to experience rapid re-epithelization, intensive proliferationof several cell types, and intensive angiogensis. In contrast, duringwound healing, down-regulation of TGF-β1 mRNA and Colα2 (I) mRNA, and adecrease in hydroxyproline levels in the wound were observed. Accordingto these results, HGF gene transfer into a skin wound aidsre-epithelization and angiogenesis, both elements of the wound healingprocess. HGF gene transfer may also suppress scar formation.

Sustained production of human HGF in the wound was confirmed at 14 daysafter gene transfer (the last day of the study) Furthermore, rat HGFconcentration in the wounds of HGF gene-transfer rats was significantlyhigher than in the control rats at three days after gene transfer.Therefore, the human HGF gene may serve as a positive regulatory factorfor the production of rat HGF. In addition, this result supports thehypothesis that HGF itself regulates local HGF production byauto-loop-positive feedback, where regulation occurs in anautocrine/paracrine manner [Kid. Int. 53: 50-58(1998); Biochem. Biophys.Res. Commun. 220: 539-545(1996)]. Thus administration of the HGF genemay promote primary HGF production in the individual receiving theadministration.

The macroscopic and histological facts yielded by the present inventionshow that when compared with control rats, HGF gene-transfer ratsexhibit hyperproliferation of basal cells in the epithelium at the edgeof a wound, and a more rapid decrease in wound lesion area. Therefore,this model shows that increased HGF mRNA and protein expression enhancesepithelial division and proliferation in the wound, and migration intoand across the wound by paracrine and/or autocrine action. In thepresent invention, an increase in the number of blood vessels andPCNA-positive staining of endothelial cells in granulation tissues wasobserved in HGF gene-transfer rats, and these findings support HGF'sstrong angiogenic action.

In this invention, semi-quantitative RT-PCR was used to observe adecrease in TGF-β1 mRNA expression in HGF gene-transfer rats, whencompared to control rats, at seven days after gene transfer.Furthermore, Colα2 (I) mRNA expression decreased at 14 days after genetransfer, and hydroxyproline decreased at 3, 7, and 14 days after genetransfer. These results suggest that scar formation in HGF gene-transferrats may be suppressed by down-regulation of TGF-β1 synthesis.Accordingly, HGF gene transfer to the skin may be useful for treatingpatients with cutaneous fibrosis.

The above-mentioned results show that gene transfer of angiogenicfactors is beneficial to the initial stages of wound healing, and thatangiogenic factors may play a role in modulating skin diseases, and thatmanipulation of re-epithelization and angiogenesis by genetransfer-induced over-expression of angiogenic factors provides a noveltherapeutic option in the field of wound healing.

1. A method for accelerating the initial stage of skin wound healing,comprising direct administration of a plasmid or viral vector encoding afull length hepatocyte growth factor (HGF) to the wound, wherein the HGFis expressed, thereby accelerating the initial stage of skin woundhealing.
 2. The method of claim 1, wherein the plasmid or viral vectorencoding a full length HGF is administered in the form of a liquidpreparation, gel, ointment, syrup, slurry, or suspension.
 3. The methodof claim 2, wherein the plasmid encoding a full length HGF isadministered in the form of HVJ-liposome.
 4. The method of claim 2,wherein the plasmid encoding a full length HGF is administered in theform of viral envelope vector.
 5. The method of claim 1, wherein theplasmid encoding a full length HUF is administered by liposomeentrapment, electrostatic liposomes, HVJ-J-liposomes, improvedHVJ-liposome, viral envelope vectors, receptor-mediated gene transfer,transfer of DNA into a cell using a particle gun (gene gun), directintroduction of naked-DNA, DNA transfer into a cell by ultrasonication,electroporation, or introduction using a positively charged polymer. 6.The method of claim 1, wherein the plasmid encoding a full length HGF isadministered in the form of HVJ-liposome.
 7. The method of claim 1,wherein the plasmid encoding a full length HGF is administered in theform of viral envelope vector.